Protein kinases, a catalyst enzyme for phosphorylation of hydroxyl groups in tyrosine, serine and threonine moieties of protein, play an important role in a growth factor signal transduction inducing growth, differentiation and proliferation on cells.
In order to maintain homeostasis in body, a signal transduction system in body should keep a balance between on and off. However, a mutation or an overexpression of particular protein kinases collapses the signal transduction system in a normal cell, e.g., by a continuous signal transduction in body, to induce various diseases such as cancer, inflammation, metabolic disease, and brain disease. Human protein kinases are estimated to exist in 518 species, about 1.7% of human whole genes (Manning et al., Science, 2002, 298, 1912), and largely divided into tyrosine protein kinases (at least 90 species) and serine/threonine protein kinases. Tyrosine protein kinases may be divided into receptor tyrosine kinases which are classified into 20 subtypes of 58 species and cytoplasm/non-receptors which are classified into 10 subtypes of 32 species. Receptor tyrosine kinases have domains on the cell surface for reception of growth factors, and active sites in the cytoplasm for phosphorylation of tyrosine moieties. When a growth factor binds to the growth factors receptor site on the cell surface of the receptor tyrosine kinase, the receptor tyrosine kinase forms a polymer and the tyrosine moieties of cytoplasm are autophosphorylated. Then, the signal transduction progresses inside nuclear by sequential phosphorylation of subfamily proteins, and finally, transcription factors inducing cancer are overexpressed.
A chromosome translocation between Bcr (breakpoint cluster region) genes located in chromosome 22 and Abl (V-abl Abelson murine leukemia viral oncogene homolog) genes located in chromosome 9 by chromosomal instability, generates oncogenes of Bcr-Abl genes. The Bcr-Abl chromosome translocation is called Philadelphia chromosome (Nowell and Hungerford, J. Natl. Cancer Inst., 1960; 25:85). In the Bcr-Abl gene, the Bcr part has oligomerization domains and the Abl part has tyrosine kinase domains. The size of the Bcr-Abl gene is determined by the cut position of the Bcr gene, and 3 subtypes (190, 210, 230 kDa) of Bcr-Abl genes have been reported. The Bcr-Abl gene is a leukemia-inducing factor, particularly p210-Bcr-Abl is a direct tumor factor inducing chronic myeloid leukemia (CML). The correlation between p210-Bcr-Abl and the CML induction is very high (>98%). Novartis developed Gleevec (imatinib mesylate) which can selectively inhibit Bcr-Abl, i.e., by inhibiting tyrosine kinases of Abl, and released in 2002. Gleevec, the first targeted antitumor agent, is being widely used as an initial standard therapy for treating CML due to its characteristic property and excellent stability. However, inactivation of Gleevec by the acquired resistance became a problem. The most important factor among various factors inducing the acquired resistance is point-mutation generated in the Abl kinase domains. There were attempts to overcome the acquired Gleevec-resistance by inhibiting such point-mutant species. Nilotinib and dasatinib, which are recently available, effectively inhibit many point-mutant species generated by the acquired Gleevec-resistance in Abl kinase domains. Among many point-mutant species generated by the acquired Gleevec-resistance in Abl kinase domains, the most important is T315I-Bcr-Abl mutant species wherein threonine 315 as a gate-keeper of Abl kinase is substituted with isoleucine. However, nilotinib and dasatinib are unable to inhibit T315I-Bcr-Abl mutant species. Accordingly, there are many attempts to develop a medicine inhibiting T315I-Bcr-Abl mutant species.
A vascular endothelial growth factors receptor (VEGFR) of a receptor tyrosine kinase (RTK) is an important modulator for angiogenesis. It is involved in generations of vascular and lymphatic vessel and in homeostasis as well as has an important effect on nerve cells. Vascular endothelial growth factors (VEGF) are predominantly produced by vascular endothelial, hematopoietic and stromal cells in response to hypoxia and upon stimulation with growth factors such as TGFs, interleukins or PDGF. VEGFs bind to VEGF receptor (VEGFR)-1, -2, and -3, and each VEGF isoform binds to a particular subset of these receptors giving rise to the formation of receptor homo- and heterodimers that activate discrete signaling pathways. Signal specificity of VEGF receptors is further modulated upon recruitment of coreceptors, such as neuropilins, heparan sulfate, integrins or cadherins.
The biological functions of VEGFs are mediated upon binding to type III RTKs, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4). VEGFRs are closely related to Fms, Kit and PDGFRs, VEGF bind to each specific receptor, VEGF-A binds to VEGFR-1 and -2 and to receptor heterodimers, while VEGF-C and -D bind VEGFR-2 and -3. P1GF and VEGF-B exclusively bind VEGFR-1 and VEGF-E interacts only with VEGFR-2. VEGF-F variants interact with either VEGFR-1 or -2. VEGF-A, -B and P1GF are predominantly required for blood vessel formation, while VEGF-C and -D are essential for the formation of lymphatic vessels. Angiogenesis provides tumors with nutrients, oxygen, and path for cancer cell spread so as to be essential for proliferation and spread. Angiogenesis in normal body is balanced by co-regulation of angiogenic stimulators and angiogenic suppressors, while in off-balanced cancer cells VEGFR is activated by growth factors (VEGF) which have a great effect on vascular endothelial cells. Various inhibitors of VEGF receptor tyrosine kinases using low molecular synthetic materials are being developed, most of which are able to be used for solid tumors and to inhibit angiogenesis activated only in cancer cells and have an excellent medicinal effect with relatively low side effects.
Tie2, a kind of receptor tyrosine kinase, is deeply concerned with angiogenesis and vasculature. The domain structure of Tie2 is conserved in all vertebrates very well (Lyons et al., Isolation of the zebrafish homologues for the tie-1 and tie-2 endothelium-specific receptor tyrosine kinases., Dev Dyn., 1998; 212:133-140). Tie2 ligands are angiopoietins (Ang). Ang2 does not induce Tie2 autophosphorylation and disturbs Tie2 activation induced by Ang1. In endothelial cells, Tie2 activation by Ang2 induces PI3K-Akt activation (Jones et al., Identification of Tek/Tie2 binding partners. Binding to a multifunctional docking site mediates cell survival and migration., J Biol Chem., 1999; 274:3089630905). In mitogen-activated protein kinases (MAPK) signal transduction path as main signal transduction system of Tie2, adapter protein GRB2 and protein tyrosine phosphatase SHP2 play an important role in dimerization process by autophosphorylation of Tie2 receptor tyrosine kinases. Ang/Tie2 and vascular endothelial growth factors (VEGF) signal transduction path perform an important function in angiogenesis of cancer cells. Tie2 is manifested in vascular endothelial, particularly in the infiltration area of cancer cells. Tie2 overexpressions are found in breast cancer (Peters et al., Expression of Tie2/Tek in breast tumour vasculature provides a new marker for evaluation of tumour angiogenesis. Br J Cancer, 1998; 77:5156) and also in uterine cancer, liver cancer, and brain cancer.
RET (rearranged during transfection), a kind of receptor tyrosine kinases is expressed mostly in nerve cells and endocrine system. N-terminal intermolecular domains of RET consist of 4 N-cadherin-like repeats, calcium-binding sites, 9 N-glycosylation sites, and cysteine-rich regions (Aiaksinen et al., Nat. Rev. Neurosci., 2002; 3:383). The cytoplasm area of RET has at least 12 tyrosine autophosphorylation sites (Liu, J., Biol. Chem., 1996; 271:5309). For example, RET9 variants have 16 autophosphorylation sites in the kinase domain. When a GFL/GFR-alpha complex binds to the intermolecular domain of RET, RET is autophosphorylated and activated (Aiaksinen et al., Nat. Rev. Neurosci., 2002; 3:383). GFL, GNDF (glial-derived neurotropic factor)-family ligands, consists of GNDF, artemin, neurturin, and persephin. GFR-alpha having 4 subtypes of GFR-alpha1-4 is known as glycosylphosphatidylinositol-anchored co-receptor. RET plays an important role in parasympathetic, enteric nervous systems, and kidney generation of mouse (Pachnis et al., Development, 1993; 119:1005). RET defunctionalization by germline mutation induce Hirschsprung's disease which is identified as a congenitial aganglionosis of distal intestines (Manie et al., Trends Genet., 2001; 17:580). However, mutations promoting the function of RET induce MEN2A (multiple endocrine neoplasia type 2A), MEN2B, and familial medullary thyroid carcinoma (FMTC). In particular, RET is proved to be a promising molecular target for development of a medicine of thyroid cancer (Cote and Gagel, N. Engl. J. Med., 2003; 349:1566).